|Year : 2017 | Volume
| Issue : 3 | Page : 421-425
Identification of carbapenemase-mediated resistance among Enterobacteriaceae bloodstream isolates: A molecular study from India
Srujana Mohanty, Mittal Gajanand, Rajni Gaind
Department of Microbiology, VMMC and Safdarjung Hospital, New Delhi, India
|Date of Web Publication||12-Oct-2017|
Department of Microbiology, VMMC and Safdarjung Hospital, New Delhi - 29
Source of Support: None, Conflict of Interest: None
Acquired resistance in carbapenem-resistant Enterobacteriaceae (CRE) conferred by carbapenemases is a major concern worldwide. Consecutive, non-duplicate isolates of Escherichia coli (EC) and Klebsiella pneumoniae from clinically diagnosed bloodstream infections were screened for the presence of carbapenem resistance by standard disk-diffusion method and minimum inhibitory concentration breakpoints using the Clinical and Laboratory Standards Institute guidelines. Carbapenemase-encoding genes were amplified by polymerase chain reaction. Of 387 isolates (214 K. pneumoniae, 173 EC) tested, 93 (24.03%) were found to be CRE. Of these, 71 (76.3%) were positive for at least one tested carbapenemase gene. The frequency of carbapenemase genes was New Delhi metallo-β-lactamse-1 (65.6%), oxacillinase (OXA)-48 (24.7%), OXA-181 (23.6%), Verona integron-encoded metallo-β-lactamase (6.4%) and K. pneumoniae carbapenemase (2.1%). Our study identified presence of carbapenemases in a large proportion of CRE isolates. Delineation of resistance mechanisms is important in view of future therapeutics concerned with the treatment of CRE and for aiding control efforts by surveillance and infection control interventions.
Keywords: Bloodstream infection, carbapenem, carbapenem-resistant Enterobacteriaceae, carbapenemase, nosocomial
|How to cite this article:|
Mohanty S, Gajanand M, Gaind R. Identification of carbapenemase-mediated resistance among Enterobacteriaceae bloodstream isolates: A molecular study from India. Indian J Med Microbiol 2017;35:421-5
|How to cite this URL:|
Mohanty S, Gajanand M, Gaind R. Identification of carbapenemase-mediated resistance among Enterobacteriaceae bloodstream isolates: A molecular study from India. Indian J Med Microbiol [serial online] 2017 [cited 2018 Oct 22];35:421-5. Available from: http://www.ijmm.org/text.asp?2017/35/3/421/216619
| ~ Introduction|| |
Nosocomial bloodstream infections (BSIs) constitute a serious health problem and cause significant morbidity and mortality.Enterobacteriaceae, especially Escherichia More Details coli (EC) and Klebsiella pneumoniae, are the topmost Gram-negative pathogens accounting for approximately 18.7%–20% and 4.7%–7.6% of bacteraemia, respectively., With the widespread increase in extended-spectrum β-lactamase (ESBL)- and AmpC-mediated β-lactamase resistance in these bacteria, carbapenems having greater stability against these enzymes were introduced as a critical reserve class of beta-lactam antimicrobials, especially in Intensive Care Units and high-risk wards., Of the different carbapenem compounds available, Imipenem (IPM), Meropenem (MEM) and Ertapenem (ETP) were US Food and Drug Administration-approved for clinical use in 1987, 1996 and 2001, respectively.,
The emergence of carbapenem-resistant Enterobacteriaceae (CRE), however, is a rapidly evolving global public health dilemma and calls for urgent action within the international scientific community., In particular, acquired resistance in Enterobacteriaceae conferred by carbapenem-hydrolysing enzymes (carbapenemases) is a major concern worldwide. An increasingly diverse range of enzymes are being recognised as significant, including serine proteases of Ambler Class A (K. pneumoniae carbapenemase [KPC]), Class B metallo-β-lactamases (New Delhi metallo-β-lactamase [NDM]; imipenemase [IMP]; Verona integron-encoded metallo-β-lactamase [VIM]) and carbapenem-hydrolysing Class D oxacillinases (OXA-48 and OXA-181).,, The corresponding genes are mostly plasmid-located and associated with various mobile genetic structures (insertion sequences, integrons, transposons), enhancing their spread. In the United States, KPC carbapenemases are more prevalent of these enzymes, while in India, other carbapenemases, in particular NDM-1, have been the most extensively observed carbapenemase in Enterobacteriaceae., Originally found in Shewanella species, OXA-48-like carbapenemases have now emerged in Enterobacteriaceae, predominantly K. pneumoniae andEC, and the level of resistance to carbapenems is usually higher when ESBL and permeability defects are associated. OXA-181, a variant of OXA-48, was initially reported in India but has been sporadically detected in the United Kingdom, The Netherlands, France, New Zealand, Oman and Singapore.,
A previous study (January–June 2008) had documented a prevalence of 10.6% and 8.8% of CRE and carbapenemase production, respectively, in our institute by phenotypic methods. Given the variation in the distribution of carbapenemases found in different geographical regions from time to time, we aimed to perform molecular identification of carbapenemase production in CRE which is the reference standard for identifying carbapenemase-producing CRE., The objectives of this study were: (i) to determine the proportion of CRE among Enterobacteriaceae bloodstream isolates, (ii) to compare the antibiogram of CRE with non-CRE isolates and (iii) to determine the distribution of selected carbapenemase-encoding genes among CRE.
| ~ Methods|| |
The study, approved by the Institutional Ethical Committee, was conducted at the Department of Microbiology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, a 1531-bedded tertiary care, teaching and referral hospital in North India over a 3-year period from January 2011 to December 2013.
Consecutive, non-duplicate isolates of EC and K. pneumoniae obtained from patients with clinically diagnosed BSIs admitted to the various wards of the hospital were included in the study. Organism identification was performed by the standard microbiological techniques, including characteristic appearance on culture media, Gram staining reaction and pattern of biochemical profiles.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing and interpretation were carried out on Mueller-Hinton agar (HiMedia, Mumbai, India) by the disk-diffusion method as per the Clinical and Laboratory Standards Institute guidelines using disks of standard concentration (HiMedia, Mumbai, India). Minimum inhibitory concentration (MIC) was determined for ETP, MEM and IPM by Etest (bioMérieux, Marcy l'Etoile, France). Standard strains of EC ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 were used as controls.
Detection of carbapenemase-encoding genes
Carbapenemase-encoding genes in the CRE isolates were amplified using a panel of primers (Sigma-Aldrich Ltd) with their expected amplicon sizes as listed in [Table 1] with single polymerase chain reactions (PCRs) for each gene.,,, Control strains used were K. pneumoniae ATCC BAA 1705 (blaKPC positive) and K. pneumoniae ATCC BAA 2156 (blaNDM positive).
|Table 1: Primers and cycling conditions used for detection of carbapenemase-encoding genes in carbapenem-resistant Enterobacteriaceae|
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| ~ Results|| |
During the study period, 387 isolates were recovered comprising of 214 K. pneumoniae (55.3%) and 173 EC (44.7%), of which 44 were from neonatal, 138 from paediatric, 128 from intensive care and 77 from adult medical units. A total of 93 (24.03%) isolates were found to be CRE (59 K. pneumoniae and 34 EC). All (100%) of CRE were ERT resistant, 65 (69.9%) MEM resistant and 51 (54.8%) IPM resistant. [Table 2] shows the overall resistance of the isolates to the antibiotics tested as well as the comparative antibiogram between the CRE and non-CRE isolates, revealing significantly higher resistance to antibiotics in CRE as compared to non-CRE isolates.
|Table 2: Comparative antimicrobial susceptibility profile of carbapenem-resistant Enterobacteriaceae and non-carbapenem-resistant Enterobacteriaceae bloodstream isolates|
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Molecular testing by PCR showed that of 93 CRE, 71 (76.3%) isolates comprising 45 K. pneumoniae and 26 EC were positive for at least one tested carbapenemase gene [Table 3]. The highest frequency was that of NDM-1 in 61 (65.6%; 39 K. pneumoniae, 22 EC), OXA-48 in 23 (24.7%, 13 K. pneumoniae, 10 EC), OXA-181 in 22 (23.6%; 18 K. pneumoniae, 4 EC), VIM in 6 (6.4%; 4 K. pneumoniae, 2 EC) and KPC in 2 (2.1%; K. pneumoniae) isolates. We did not find blaIMP gene in any of the isolates. Forty-three isolates harboured a single carbapenemase gene (NDM-1 in 39, KPC in 2, OXA-48 in 1 and OXA-181 in 1), while 28 (39.4%) isolates co-harboured more than one carbapenemase gene. Of the latter, 17 isolates co-harboured two carbapenemase genes (NDM-1 and OXA-181, NDM-1 and OXA-48, OXA-48 and OXA-181, VIM and OXA-48 in 6, 5, 4 and 2 isolates, respectively), 8 isolates co-harboured three carbapenemase genes (NDM-1, OXA-48 and OXA-181 in 7 and NDM, OXA-48 and VIM in 1 isolate) and 3 isolates co-harboured four carbapenemase genes (NDM-1, VIM, OXA-48 and OXA-181) [Table 3]. The rate of carbapenemase production was observed to be the same in carbapenem-resistant K. pneumoniae (45/59, 76.3%) and EC (26/34, 76.4%) isolates.
|Table 3: Distribution of carbapenemase genes according to carbapenem minimum inhibitory concentration in carbapenem-resistant Enterobacteriaceae (n=93)|
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| ~ Discussion|| |
The earliest reports of carbapenemase-mediated resistance in Enterobacteriaceae from India consist of NDM-1 producing Enterobacteriaceae from Mumbai in 2010 and dissemination of both NDM-1 and OXA-181 producing Enterobacteriaceae collected from various Indian hospitals during 2006 and 2007. In the former study, of 24 CRE recovered from a tertiary hospital, 22 were found to be harbouring NDM-1 gene (10 were Klebsiella spp., 9 EC, 2 Enterobacter spp. and 1 Morganella morganii). In the latter study, among 39 CRE (2.7% overall; EC, Enterobacter cloacae and K. pneumoniae strains) isolated in 2006 and 2007 in India, 15 strains carried blaNDM-1 and 10 harboured blaOXA-181. Subsequent reports revealed an increasing prevalence and dissemination of NDM-1β-lactamase, often in conjunction with other carbapenemase genes such as blaOXA-48, blaKPC and blaVIM in various combinations,,, as has been observed in the present study.
The current study (2011–2013) showed a prevalence of 24.03% carbapenem resistance amongst Enterobacteriaceae bloodstream isolates in our institute, which is more than twice the prevalence (10.6%) compared to our earlier study performed 3 years prior in 2008. Similarly, a study on neonatal septicaemia has reported an increase in CRE from 11% in 2008 to 37.5% in 2011. Elsewhere, the prevalence of carbapenem-resistant K. pneumoniae has been found to vary from 4.05% in Malaysia to 5%, 8% and 13% in Argentina, USA and Greece, respectively.
Overall, carbapenemase-mediated resistance was identified in 71 (76.3%) CRE isolates with the highest prevalence conferred by NDM-1 genes, followed by OXA-48 and OXA-181 in our setting. Co-occurrence of multiple carbapenemase genes was found in 39.4% isolates. In contrast, in a recent study conducted by the Centers for Disease Control and Prevention, USA, on a collection of CRE isolates (January 2011–January 2014) from different participating centres, only 30% (94/312) of CRE had carbapenemase genes with majority as KPC (93%–100%) and few as NDM (6%–7%). However, another study during the same time period in Los Angeles, California, from 2011 to 2013 showed a higher proportion of carbapenemase-encoding genes in CRE (81.7%, 94/115) and included blaKPC(78.3%), blaNDM-1 (0.9%) and blaSME(2.6%). Elsewhere, the proportion of carbapenemase-encoding genes in CRE have been reported to vary from 60.8% in China to 77.3% in Sultanate of Oman and 98% in Turkey, with multiple genes detected in 10%, 5.9% and 13% of the carbapenemase-positive isolates, respectively. The predominant genes have been KPC-2 (54.9%), NDM (50%) and OXA-48 (86%) in these studies.
Imipenem (MIC50 1.5 μg/ml) had better activity than meropenem (MIC50 3 μg/ml) against ertapenem-non-susceptible Enterobacteriaceae isolates in the current study with 65 (69.9%) MEM-resistant compared to 51 (54.8%) IPM-resistant isolates. However, susceptibility to both MEM and IPM was uniformly retained in isolates with ERT MIC ≤4 μg/ml, but, with an increase in ERT MIC ≥6 μg/ml, most of the EC and K. pneumoniae were synchronously resistant to MEM and IPM [Table 3]. A similar finding had been observed by Jean et al., where E. coli and K. pneumoniae with ETP MIC ≥4 μg/ml were synchronously resistant to MEM and IPM. These data provide a better understanding of choosing appropriate carbapenem agents to treat infections caused by carbapenem-non-susceptible Enterobacteriaceae isolates.
| ~ Conclusion|| |
Our study identified the presence of carbapenemase-mediated resistance in a large proportion of CRE isolates. Delineation of resistance mechanisms is important in view of future therapeutics concerned with the treatment of CRE (e.g. using carbapenems in combination with carbapenemase-inhibitors for the treatment of carbapenemase-producing isolates) and for aiding control efforts by surveillance and infection control interventions.
Financial support and sponsorship
This work was supported by the Indian Council of Medical Research, Government of India, through an extramural research grant (5/3/10/2010-ECD-1).
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Correa L, Pittet D. Problems and solutions in hospital-acquired bacteraemia. J Hosp Infect 2000;46:89-95.
Pfaller MA, Jones RN, Doern GV, Kugler K. Bacterial pathogens isolated from patients with bloodstream infection: Frequencies of occurrence and antimicrobial susceptibility patterns from the SENTRY antimicrobial surveillance program (United States and Canada, 1997). Antimicrob Agents Chemother 1998;42:1762-70.
Livermore DM, Hope R, Brick G, Lillie M, Reynolds R; BSAC Working Parties on Resistance Surveillance. Non-susceptibility trends among Enterobacteriaceae
from bacteraemias in the UK and Ireland, 2001-06. J Antimicrob Chemother 2008;62 Suppl 2:ii41-54.
Lincopan N. Metallo-β-lactamase producing Klebsiella pneumoniae
. In: Khan AU, editor. Current Trends in Antibiotic Resistance in Infectious Diseases. New Delhi, India: IK International Publishing House Pvt. Ltd.; 2009. p. 136-64.
Lister PD. Carbapenems in the USA: Focus on doripenem. Expert Rev Anti Infect Ther 2007;5:793-809.
Centers for Disease Control and Prevention (CDC). Vital signs: Carbapenem-resistant Enterobacteriaceae
. MMWR Morb Mortal Wkly Rep 2013;62:165-70.
Bush K. Proliferation and significance of clinically relevant ß-lactamases. Ann N
Y Acad Sci 2013;1277:84-90.
Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae
: Here is the storm! Trends Mol Med 2012;18:263-72.
Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: The phantom menace. J Antimicrob Chemother 2012;67:1597-606.
Castanheira M, Deshpande LM, Mathai D, Bell JM, Jones RN, Mendes RE. Early dissemination of NDM-1-and OXA-181-producing Enterobacteriaceae
in Indian hospitals: Report from the SENTRY Antimicrobial Surveillance Program, 2006-2007. Antimicrob Agents Chemother 2011;55:1274-8.
Balm MN, Ngan G, Jureen R, Lin RT, Teo JW. OXA-181-producing Klebsiella pneumoniae
establishing in Singapore. BMC Infect Dis 2013;13:58.
Mohanty S, Gaind R, Ranjan R, Deb M. Prevalence and phenotypic characterisation of carbapenem resistance in Enterobacteriaceae
bloodstream isolates in a tertiary care hospital in India. Int J Antimicrob Agents 2011;37:273-5.
Collee JG, Miles RS, Watt B. Tests for the identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th
ed. London: Churchill Livingstone; 1996. p. 131-45.
CLSI Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 22nd
Informational Supplement. CLSI Document M100-S22. Wyne, PA: Clinical and Laboratory Standards Institute; 2012.
Kothari C, Gaind R, Singh LC, Sinha A, Kumari V, Arya S, et al.
Community acquisition of β-lactamase producing Enterobacteriaceae
in neonatal gut. BMC Microbiol 2013;13:136.
Siu LK, Huang DB, Chiang T. Plasmid transferability of KPC into a virulent K2 serotype Klebsiella pneumoniae
. BMC Infect Dis 2014;14:176.
Miriagou V, Tzelepi E, Gianneli D, Tzouvelekis LS. Escherichia coli
with a self-transferable, multiresistant plasmid coding for metallo-beta-lactamase VIM-1. Antimicrob Agents Chemother 2003;47:395-7.
Shanthi M, Sekar U, K A, Bramhne HG. OXA-181 beta lactamase is not a major mediator of carbapenem resistance in Enterobacteriaceae
. J Clin Diagn Res 2013;7:1986-8.
Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hedge A, Soman R, et al.
New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae
: Treatment options with carbapenems compromised. J Assoc Physicians India 2010;58:147-9.
Lascols C, Hackel M, Marshall SH, Hujer AM, Bouchillon S, Badal R, et al.
Increasing prevalence and dissemination of NDM-1 metallo-ß-lactamase in India: Data from the SMART study (2009). J Antimicrob Chemother 2011;66:1992-7.
Bora A, Ahmed GU, Hazarika NK, Prasad KN, Shukla SK, Randhawa V, et al.
Incidence of bla NDM-1 gene in Escherichia coli
isolates at a tertiary care referral hospital in Northeast India. Indian J Med Microbiol 2013;31:250-6.
] [Full text]
Kazi M, Drego L, Nikam C, Ajbani K, Soman R, Shetty A, et al.
Molecular characterization of carbapenem-resistant Enterobacteriaceae
at a tertiary care laboratory in Mumbai. Eur J Clin Microbiol Infect Dis 2015;34:467-72.
Datta S, Roy S, Chatterjee S, Saha A, Sen B, Pal T, et al.
Afive-year experience of carbapenem resistance in Enterobacteriaceae
causing neonatal septicaemia: Predominance of NDM-1. PLoS One 2014;9:e112101.
Hamzan NI, Yean CY, Rahman RA, Hasan H, Rahman ZA. Detection of blaIMP4 and blaNDM1 harboring Klebsiella pneumoniae
isolates in a university hospital in Malaysia. Emerg Health Threats J 2015;8:26011.
Chea N, Bulens SN, Kongphet-Tran T, Lynfield R, Shaw KM, Vagnone PS, et al.
Improved phenotype-based definition for identifying carbapenemase producers among carbapenem-resistant Enterobacteriaceae
. Emerg Infect Dis 2015;21:1611-6.
Pollett S, Miller S, Hindler J, Uslan D, Carvalho M, Humphries RM. Phenotypic and molecular characteristics of carbapenem-resistant Enterobacteriaceae
in a Health Care System in Los Angeles, California, from 2011 to 2013. J Clin Microbiol 2014;52:4003-9.
Hu L, Zhong Q, Shang Y, Wang H, Ning C, Li Y, et al.
The prevalence of carbapenemase genes and plasmid-mediated quinolone resistance determinants in carbapenem-resistant Enterobacteriaceae
from five teaching hospitals in central China. Epidemiol Infect 2014;142:1972-7.
Dortet L, Poirel L, Al Yaqoubi F, Nordmann P. NDM-1, OXA-48 and OXA-181 carbapenemase-producing Enterobacteriaceae
in Sultanate of Oman. Clin Microbiol Infect 2012;18:E144-8.
Iraz M, Özad Düzgün A, Sandallı C, Doymaz MZ, Akkoyunlu Y, Saral A, et al.
Distribution of β-lactamase genes among carbapenem-resistant Klebsiella pneumoniae
strains isolated from patients in turkey. Ann Lab Med 2015;35:595-601.
Jean SS, Hsueh PR, Lee WS, Yu KW, Liao CH, Chang FY, et al.
Carbapenem susceptibilities and non-susceptibility concordance to different carbapenems amongst clinically important Gram-negative bacteria isolated from Intensive Care Units in Taiwan: Results from the Surveillance of Multicentre Antimicrobial Resistance in Taiwan (SMART) in 2009. Int J Antimicrob Agents 2013;41:457-62.
[Table 1], [Table 2], [Table 3]